Competitive binding assays are established analytical methods in medicinal and clinical chemistry. A typical competitive binding assay constitutes a receptor and a signaling unit that also serves as a surrogate substrate. The signaling unit possesses an easily observable and quantifiable property, which is modulated in response to competitive binding with an analyte. For instance, modulation of absorbance or emission, or the ability to catalyze a reaction, are common approaches. When the signaling unit is a pH or solvatochromic indicator, the assay is specifically called an indicator-displacement assay.
The advantages of an indicator-displacement assay include: 1) the need to covalently incorporate the chromophore or fluorophore into the structures of receptors or analytes is eliminated; 2) the indicators are exchangeable; 3) the detection mechanism is not directly perturbed by the analyte structures; and 4) secondary tuning of sensitivity and selectivity is available because of the participation of the indicator. The usual molecular recognition driving forces are exploited in constructing successful indicator-displacement assays. So far, assays utilizing ion pairing, hydrogen bonding, reversible covalent interactions, metal coordination, and combinations of these, have been documented. Many physiologically and environmentally important targets, such as phosphate, pyrophosphate, citrate, carbonate, amino acids, etc., can now be detected and quantified through indicator-displacement assays. Despite the successes, the available applications of indicator-displacement assays have been limited to sensing the identity and quantity of given analytes. Indicator-displacement assays have been used in a number of sensing applications, but not in quantification of enantiomeric excess (ee) of a chiral analyte.
However, there is a growing demand for methods of ee determination. For example, the FDA currently requires that pharmaceutical companies create enantiomerically pure substances, or that the enantiomer of the drug be thoroughly studied and found to have no adverse side effects. The synthesis of enantiomerically pure substances requires the use of reagents that produce an enantiomeric excess (ee) of the desired drug enantiomer or its chemical precursor. High throughput screening for such enantioselective reagents entails product-analyzing assays for rapid determination of both the yields and ees from given catalytic reactions. This double-parameter requirement increases the difficulty of assay design. Traditional enantioselective optical chemosensors, such as BINOL based compounds (−)-6 and 7 in FIG. 10, usually rely upon cumbersome empirical ee calibration curves against absorbance or fluorescence intensity for each total concentration of the chiral analyte. Thus a need exists for assays capable of rapidly and accurately determining the concentrations and ees of chiral samples.